Naphthyridinomycin (I), a novel isoquinoline quinone antitumor antibiotic produced by Streptomyces lusitanus, was previously demonstrated to be derived from tyrosine, the S-methyl group of methionine, ornithine, and glycine. However, whether the incorporation of carbons 1 and 2 of glycine into naphthyridinomycin's C1 and C2 was direct or indirect (via metabolism) was undetermined. In this study, labeled precursors ([1,2-13C]glycine, [2-13C]glycine, L-[3-13C]serine) were fed to S. lusitanus on day three of fermentation, and cyanonaphthyridinomycin was isolated and analyzed by 13C NMR. Results showed that serine is a more immediate precursor for carbons 1 and 2 of naphthyridinomycin than glycine, as glycine is first converted to serine prior to incorporation. L-[3-13C]serine enriched carbon 2 of the antibiotic, while L-[1-14C]serine did not label the antibiotic (＞0.1% incorporation), indicating the loss of C1 of serine. These results support the conclusion that serine is a more immediate precursor for carbons 1 and 2 of naphthyridinomycin than glycine. The origin of the nitrogen at C1 (from serine or the delta amino group of ornithine) is currently under investigation.